The present invention relates to the structure and function of the polo-box of the mitotic protein polo kinase gene, to proteins that bind to the polo box and their encoding genes, and particularly to compositions and methods for modulation of polo box function and regulation of mitotic processes for treatment of a subject having unwanted eukaryotic cells such as cancer, fungal pathogens, pathogenic protozoans, helminths, and arthropod cells.
Polo kinases are found in diverse eukaryotic organisms, as represented by mammalian Plk, amphibial Xenopus laevis Plx1, and others shown in FIG. 1, and share a region of sequence homology in the carboxy terminal (C-terminal) non-catalytic domain termed the polo-box (Clay, F. J. et al., 1993, Proc. Natl. Acad. Sci. USA 90: 4882-4886). Genetic and biochemical analyses show that polo kinases affect diverse cellular events during M phase such as centrosome maturation (Lane, H. A. et al., 1996, J. Cell Biol, 135: 1701-1713) and bipolar spindle formation (Llamazares, S., et al., 1991, Genes and Development, 5: 2153-2165; Ohkura, H., et al., 1995, Genes and Development, 9: 1059-1073. Polo kinases appear to affect steps in the transition from G2 to M (G2/M) phase, such as activation of Cdc2 through Cdc25C phosphatase (Kumagai, A., et al., 1996, Science, 273: 1377-1380), DNA damage checkpoint adaptation (Toczyski, D. P. et al., 1997, Cell, 90: 1097-1106), and the anaphase-promoting complex (Shirayama, M. et al., 1998, EMBO J, 17: 1336-1349); Descombes et al., 1998, EMBO J, 17: 1328-1335). Expression of Plo1 or Plk has been shown to induce cytokinesis-associated septation structures (Ohkura, H., et al., 1995, Genes and Development, 9: 1059-1073).
The exact structure and the function of the conserved sequence polo box in the non-catalytic domain polo-box are undefined. Regulatory sites in other regions of the gene have not been defined. This no information on cellular elements such as proteins that interact with the polo kinase. Regulation of polo kinase activity and the role of the polo kinase protein in regulation of events of mitosis are not understood.
An embodiment of the invention is a composition having sufficient affinity for a polo-box of a polo kinase so as to inhibit localization of a polo kinase in a cell. The composition can be a peptide or a peptide analog, for example, a peptide is selected from the group consisting of an amino acid sequence from a tubulin, an actin, a chaperonin, a Golgi protein, and a translationally controlled tumor protein, or it can comprise all or a portion of a carboxy terminal of a polo kinase. Further, the composition having sufficient affinity for a polo-box of a polo kinase can be a peptidomimetic.
An embodiment of the invention is a composition which is a peptide amino acid sequence comprising all or a portion of an amino acid consensus sequence in the carboxy terminal of polo kinase proteins of eukaryotic organisms, the consensus sequence being located between and not including the polo box PB1 (SEQ ID NO: 1) and the carboxy terminal. The composition of this embodiment is selected from the group consisting of amino acid sequences comprising all or a portion of:
NH2-ser-X-X-pro-X-ser-leu-X-X-lys-X-X-leu-leu-X-X-phe-X-X-tyr-met-X-X-X-leu-X-lys-ala-COOH (PB2; SEQ ID NO:6), and
NH2-leu-X-X-X-X-arg-thr-X-X-ala-X-X-X-X-leu-ser-asn-gly-X-X-gln-X-asn-X-phe-X-asp-his-X-lys-COOH (PB3; SEQ ID NO:7), wherein X in a position denotes an unspecified amino acid residue. Further, the composition can be a polynucleotide selected from the group consisting of: a polynucleotide encoding an amino acid sequence according PB2 and PB3 (SEQ ID Nos:6 and 7), and a polynucleotide complementary to such a polynucleotide. The composition which is a peptide of amino acid sequence according to these polo box consensus sequences, can be obtained from a target cell selected from the group of cells consisting of a fungus, a protozoan, an arthropod, a helminth, and a tumor cell of a mammal, so that in the amino acid sequence the amino acid residues denoted X are obtained from the amino acid sequence of a polo kinase in a cell which is an unwanted cell in a subject.
Another embodiment of the invention is a method of inhibiting growth of an unwanted cell in a subject by introducing into the cell a polo-box binding inhibitor, for example, a peptide or peptide analog comprising an amino acid sequence of a polo kinase, for example, the amino acid sequence from the carboxy terminal of the polo kinase. In this embodiment of the invention, the unwanted cell in the subject can be a cancer cell, for example, a cancer of a lung, a breast, a uterus, an ovary, a cervix, an epithelium, a brain, a retina, a prostate, and a throat. Further, the unwanted cell in the subject can be a cell of a fungus, a protozoan, an arthropod, and a helminth.
Another embodiment of the invention is a method of inhibiting cell division of an unwanted cell by modulation of a function of a polo kinase in a cell, for example, modulation of localization of a polo kinase during a mitotic phase of a cell.
An embodiment of the invention is a method for screening for an agent that modulates activity of a polo kinase comprising: culturing under permissive and restrictive growth conditions a cell, the cell having a conditional lethal endogenous chromosomal polo kinase gene complemented by a recombinant vector encoding a regulatable polo kinase gene such that growth of the cell at the restrictive condition is dependent on expressing the regulatable gene; inducing expression of the regulatable polo kinase gene; and exposing a portion of the culture of the cell to a candidate agent under the permissive and the restrictive growth condition, so that inhibition of growth of the cell in the presence but not the absence of the candidate inhibitor at the restrictive growth condition is an indication that the candidate inhibitor is an agent that modulates the activity of the polo kinase. In this embodiment, the polo kinase can be one that is selected from the group of consisting of a polo kinase from a fungus, a protozoan, an arthropod, a helminth, and a tumor cell. This method can comprise an additional step of testing the candidate inhibitor in a pharmaceutically acceptable carrier for appropriate dose in preclinical testing with an animal model system, for example, an animal infected with a fungus. The fungal infection can be selected from the group consisting of aspergillosis, candidiasis, a Lichen planus infection, and athlete""s foot.
Another embodiment of the invention is a method for obtaining a DNA sequence encoding a peptide that binds to a polo box peptide, comprising: constructing a two-hybrid system wherein the bait is a peptide having an amino acid sequence comprising a polo box and the prey is is a cDNA library, the binding of the two hybrid proteins of the bait and the potential prey causing a change in a selectible phenotype of the cell; transforming a population of the cells with the constructed two-hybrid vector carry the library of donor DNA sequences; growing the cells under conditions that induce expression of the two-hybrid fusion genes and the appearance of the selectible phenotype in cells carrying a DNA sequence encoding a potential prey peptide that binds to a polo box peptide; and selecting cells expressing the selectible phenotype to isolate a cell clone carrying a DNA sequence from the library, such that the translation product of the cloned DNA binds to the polo box of the polo kinase. This method can have the additional step of sequencing the cloned DNA. Further, this method can have the additional step of isolating the gene from the donor organism comprising the cloned DNA encoding the prey. From this method that is an embodiment of the invention can be cloned a cortical actin, a septin, a tubulin, a cytoplasmic chaperonin complex protein, a Golgi protein, and a translationally controlled tumor protein, for example, a peptide of a protein selected from the group consisting of: a polo kinase C-terminal, a Grasp 65 (SEQ ID No:24), a chaperonin-containing-TCP (CCT-xcex5), and a translationally controlled tumor protein p23 (TCTP/p23) (SEQ ID No:27). Further, this method can comprise an additional step of determining an effective dose of the modulator for a subject having an unwanted cell which is selected from the group of cells consisting of a human cancer cell, an arthropod cell, and a fungal cell. By use of the method of inhibiting growth of an unwanted cell in a subject, one can administer to the subject an effective dose of the modulator obtained, in a pharmaceutically acceptable carrier.
An embodiment of the invention provides a method of developing an organism-specific polo box binding inhibitor, comprising: providing a sequence of a polo kinase from the organism; identifying the organism""s polo boxes in the polo kinase from the organism by aligning the sequence of the polo kinase from the organism with each of PB1 (SEQ ID NO:1), PB2 (SEQ ID NO:6) and PB3 (SEQ ID NO:7) and using all or a portion of the organism""s polo boxes to develope a polo box binding inhibitor.